DNA-binding determinants promoting NHEJ by human Polμ.

Abstract

Non-homologous end-joining (NHEJ), the preferred pathway to repair double-strand breaks (DSBs) in higher eukaryotes, relies on a collection of molecular tools to process the broken ends, including specific DNA polymerases. Among them, Polµ is unique as it can catalyze DNA synthesis upon connection of two non-complementary ends. Here, we demonstrate that this capacity is intrinsic to Polµ, not conferred by other NHEJ factors. To understand the molecular determinants of its specific function in NHEJ, the interaction of human Polµ with DNA has been directly visualized by electromobility shift assay and footprinting assays. Stable interaction with a DNA gap requires the presence of a recessive 5'-P, thus orienting the catalytic domain for primer and nucleotide binding. Accordingly, recognition of the 5'-P is crucial to align the two DNA substrates of the NHEJ reaction. Site-directed mutagenesis demonstrates the relevance of three specific residues (Lys(249), Arg(253) and Arg(416)) in stabilizing the primer strand during end synapsis, allowing a range of microhomology-induced distortions beneficial for NHEJ. Moreover, our results suggest that the Polµ BRCT domain, thought to be exclusively involved in interaction with NHEJ core factors, has a direct role in binding the DNA region neighbor to the 5'-P, thus boosting Polµ-mediated NHEJ reactions.

DNA-binding properties of human family X polymerases: importance of the 5′-P group for DNA binding. (A) Scheme of the substrates used for the footprinting assays. To produce this substrate the oligonucleotides FP-T (template), FP-P (primer) and FP-D (downstream) were hybridized. (B) DNA-binding affinity of the indicated proteins (Polβ and Polµ at 100 nM, Polλ at 500 nM) was assayed as described in ‘Materials and Methods’ section, using the footprinting substrate radioactively labeled at the 5′-end of the template strand. Gel was dried and the labeled fragments detected by autoradiography. (C) Footprinting assay of the control protein Spo0A (1 µg) or each of the members of the X family (Polβ, 1.5 µg; Polµ, 1.5 µg; Polλ, 10 µg, TdT, 10 µg) was conducted as described in ‘Materials and Methods’ section, in the presence of 100 µM dTTP and 2.5 mM MgCl2. Ten micrograms of BSA were added to the control lane. (D) EMSA of Polβ and Polµ (100 nM) using a gapped DNA substrate formed by the oligonucleotides Sp1C (labeled at the 5′-end), T28 and D12, the latter either having (P) of lacking (OH) a 5′-P group. Gel was dried and the labeled fragments detected by autoradiography. E) Footprinting assay of Polβ (1.5 µg) or Polµ (1.5 µg) with a gapped substrate that either contains (P) or lacks (OH) a 5′-P group in the downstream strand, in the presence of 100 µM dTTP and 2.5 mM MgCl2. Ten micrograms of BSA was added to the control lane. Gel was dried and the labeled fragments detected by autoradiography.

Ternary complex formation in solution. (A) Structure of Polµ (shown in wheat-colored ribbons) ternary complex (2IHM), in which the DNA substrate and incoming nucleotide are shown in sticks with the following colors: dNTP, dark teal; template strand, green; primer strand, yellow; downstream strand, dark pink. Selected residues are shown in red sticks. Nucleotides in the template strand are numbered as in the footprinting assays for clarity. (B) Footprinting assay of the wild-type Polβ (1.5 µg) and Polµ (1.5 µg). When indicated, 100 µM dATP or dTTP were added, together with 2.5 mM MgCl2. The DNA substrate used, formed by hybridizing the oligonucleotides FP-T (template, labeled at its 5′-end), FP-P (primer) and FP-D (downstream) always contains a phosphate group at the 5′-end of the downstream strand. Gel was dried and the labeled fragments detected by autoradiography. (C) Footprinting assays of Polµ mutants H329G and R387K (1.5 µg) were carried out as described in (B).

Intrinsic Polµ binding to NHEJ substrates. (A) EMSA of Polµ (300 and 600 nM) using T/D substrates (formed by hybridization of GT and NHEJ-D oligonucleotides) either lacking (OH) or having (P) a phosphate group at the 5′-end of the downstream strand. The schemes show the free substrate (with a sphere depicting the 5′-P group) or the polymerase bound to the substrate. (B) Schematic representation of the possible arrangement of the three NHEJ substrates (the two DNA ends and the incoming nucleotide) mediated by Polµ.

Impact of Polµ DNA-binding properties on its enzymatic activity during NHEJ. (A) Gap-filling activity of Polβ, Polλ and Polµ (25 nM each) was assayed using a substrate formed by the hybridization of the oligonucleotides SP1C, T28 and D12. When indicated, 10 nM of each dNTP was added, in the presence of 2.5 mM MgCl2. (B) NHEJ assay of Polβ (600 nM), Polλ (600 nM) and Polµ (200 nM) was performed as described in ‘Materials and Methods’ section, using a set of compatible substrates: the labeled substrate was formed by hybridization of GT and NHEJ-D (shown in light gray) and the cold substrate by hybridization of CA and NHEJ-D (shown in dark gray). When indicated, dNTPs were added separately at 100 µM in the presence of 1 mM MnCl2 for Polβ and Polλ, and 2.5 mM MgCl2 for Polµ. After electrophoresis, the labeled fragments were detected by autoradiography. (C) NHEJ reaction performed as in (B), with a set of incompatible substrates in which both the labeled (light gray) and cold (dark gray) molecules were formed by hybridization of C- and D-NHEJ. When indicated, the substrates contain a 5′-P group at the downstream strand (dark gray spheres).

Importance of 5′-P recognition for Polµ-mediated NHEJ. (A) NHEJ assay of Polµ (200 nM) performed as described in ‘Materials and Methods’ section, using a set of compatible substrates: the labeled substrate was formed by hybridization of GT and NHEJ-D (light gray) and the cold substrate by hybridization of CA and NHEJ-D (dark gray). The dark gray spheres indicate the presence of a 5′-P group in the downstream strand of the substrate. When indicated, dNTPs were added separately at 1 µM in the presence of 2.5 mM MgCl2. After electrophoresis, the labeled fragments were detected by autoradiography. (B) NHEJ reaction performed as in (C), with a set of incompatible substrates in which both the labeled (light gray) and cold (dark gray) molecules were formed by hybridization of C and NHEJ-D.

NHEJ-specific Polµ residues acting as ligands of the priming end. (A) Cartoon representation of the ternary complex structure of Polµ (PDB ID: 2IHM), in which the DNA substrate has been modified to mimic a 1-nt 3′-protruding substrate in a template/primer orientation, colored light/dark blue. The incoming dNTP is shown in green. The residues selected for mutagenesis are shown in red sticks, and the polar contacts established with the primer strand are highlighted in black. (B) Sequence alignment of the four X family members in humans, showing the two regions in which the mutations were made. Numbering indicates the residues in the Polµ sequence. Mutated residues are indicated with gray dots. (C) Gap-filling reactions were performed as described in ‘Materials and Methods’ section with the indicated proteins (25 nM) using a gapped substrate containing the oligonucleotides SP1C, T28 and D12. When indicated, dNTPs were added separately at 10 nM in the presence of 2.5 mM MgCl2. (D) Terminal transferase activity assay with the indicated proteins (600 nM) using a homopolymeric substrate (polydA) and each of the four dNTPs (100 µM). Reactions were performed for 30 min at 37°C. The insertion of dTTP which can be still observed with the mutants is not a strict terminal transferase reaction, but the result of connecting two ssDNA substrates and subsequently copying the polydA template. (E) NHEJ reactions were performed with 200 nM of the indicated proteins and using four sets of substrates: the labeled substrates were formed by hybridization of C with NHEJ-D or D3 with D1, and the cold substrates, by hybridization of either C with NHEJ-D or D4 with D2. The dark gray spheres indicate the presence of a 5′-P group in the downstream strand of the substrate. When indicated, each of the four ddNTPs (10 µM) were added in the presence of 2.5 mM MgCl2. (F) Gap-filling reactions were performed as in (C) but using a 2-nt gapped substrate containing the oligonucleotides P15, T32 and D16. When indicated, the dNTP complementary to the first (dCTP), second (dGTP) or both templating bases were added (10 nM). (G) NHEJ reactions were performed as in (E) but using one set of substrates that gives shape to a 2-nt gap once joined: the labeled substrate contained the oligonucleotides D3 and D1, and the cold substrate, D4–AC and D2. The dark gray spheres indicate the presence of a 5′-P group in the downstream strand of the substrate. When indicated, each of the four dNTPs (10 µM) was added in the presence of 2.5 mM MgCl2.

Polµ-mediated NHEJ: non-aligned ends. (A) NHEJ reactions performed as described in ‘Materials and Methods’ section, with 200 nM Polµ and using labeled compatible substrates: the short, fast running substrates were formed by hybridization of either D3 (second and fourth schemes), D3BB1 (first scheme) or D3BB2 (third scheme) with D1 (shown in light gray) and the long, slow running substrates, by hybridization of either D4 (first and third schemes), D4BB1 (second scheme) or D4BB2 (fourth scheme) with D2 (shown in dark gray). The dark gray spheres indicate the presence of a 5′-P group in the downstream strand of the substrate. When indicated, dNTPs were added separately at 1 µM in the presence of 2.5 mM MgCl2. After electrophoresis, the labeled fragments were detected by autoradiography. (B) NHEJ reactions performed as in (A), using labeled compatible substrates: the short substrates were formed by hybridization of either D3 (second and fourth schemes), D3MM (first scheme) or D3FLAP (third scheme) with D1 (shown in dark gray) and the long substrates, by hybridization of either D4 (first to third schemes) or D4FLAP (fourth scheme) or D4BB2 (fourth scheme) with D2 (shown in dark gray). The dark gray spheres indicate the presence of a 5′-P group in the downstream strand of the substrate.

The BRCT domain of Polµ contributes, via DNA binding, to its intrinsic ability of joining DNA ends. (A) Gap-filling reactions were performed as described in ‘Materials and Methods’ section for the indicated proteins (25 nM) using a gapped substrate containing the oligonucleotides SP1C, T28 and D12. When indicated, dNTPs were added separately at 10 nM in the presence of 2.5 mM MgCl2. (B) Footprinting assay of the wild-type Polβ (1.5 µg) and mutant and wild-type Polµ (1.5 µg). The DNA substrate used, formed by hybridization of the oligonucleotides FP-T (template, labeled at its 5′-end), FP-P (primer) and FP-D (downstream) may have (P) or lack (OH) a phosphate group at the 5′-end of the downstream strand. (C) NHEJ reactions were performed as described in ‘Materials and Methods’ section, with 200 nM Polµ and using two sets of substrates: the labeled substrates were formed by hybridization of GT or C with NHEJ-D, and the cold substrates, by hybridization of either CA or C with NHEJ-D. The dark gray spheres indicate the presence of a 5′-P group in the downstream strand of the substrate. When indicated, dNTPs were added separately at 10 µM in the presence of 2.5 mM MgCl2. The efficiency of the polymerization reaction is defined as a function of the percentage of primer extension. We used two different software programs to obtain the measurement of the extended primer in a minimum of three different replica experiments. (D) EMSA was performed with the indicated proteins (300 and 600 nM) using a 3′-protruding substrate containing the oligonucleotides GT and NHEJ-D.

Model of the interaction of Polµ with the Ku heterodimer and the DNA substrate through the BRCT domain. Computer-generated model of the predicted location of the Polµ β-like core, the BRCT domain and the Ku70/80 heterodimer on a single DNA end. The different domains in the Polµ core have been colored as follows: 8-kDa domain in green (with the residues forming the 5′-P pocket highlighted in dark green), the fingers subdomain in yellow, the palm subdomain in salmon, the thumb subdomain in pink and the Loop1 motif in cyan. The BRCT domain is colored light blue, with the residues predicted to be interacting with the DNA substrate in dark blue and the residues implicated in the interaction with the NHEJ factors in red. The two subunits of the Ku heterodimer are colored in dark and light orange.